T and B cell Isolation

Reagents Heparin - 1000 U/ml Ficoll-Hypaque PBS RPMI-1640 supplemented with 10 mM glutamine and 15% FBS AET (0.14M) Dissolve 1.967 g AET in 35 ml di-H2O. Adjust to pH 8.0 with 1.0N NaOH. Bring volume to 50 ml with di-H2O. Store at 2-8oC. Check pH every 2 weeks. AET-treated SRBC Wash SRBC 4 times with PBS  Add 4 volumes AET to 1 volume packed SRBC in a 15 m conical tube (1 ml of AET + 0.25 ml packed SRBC).  Mix well. Incubate in a 37oC water bath for 30 minutes. Shake vigorously.  Wash 3 times with PBS.  Store in PBS at 2-8oC for up to 3 days. SRBC-Absorbed FBS Mix 10 volumes of FBS with 1 volume packed SRBC.  Incubate at 37oC fir 30 minutes.  Incubate at 2-8oC for 30 minutes.  Centrifuge at 400 g for 10 minutes.  Collect the FBS. Filter sterilize. Store aliquots at -20oC. Preparation of PBL's Draw peripheral blood into syringe containing 10 U/ml heparin. Dilute the blood 1:1 with PBS. Layer 30 ml of diluted blood onto 20 ml Ficoll-Hypaque.  Centrifuge at 1550 rpm for 30 minutes, room temperature.  Aspirate and discard the supernatant.  Carefully collect the interface of PBL's and transfer into a clean tube.  Fill the tube with PBS. Centrifuge at 1550 rpm for 10 minutes.  Wash the pellet 2 times with PBS.  Count the cells and resuspend to 107 cells/ml in PBS. Separation of T-Cells Mix 1 ml of AET-treated SRBC with 10 ml FBS. Mix and equal volume of PBL's with a 1% (v/v) mixture of AET-SRBC_FBS in a 50 ml tube. Incubate in a 37oC water bath for 10 minutes.  Centrifuge at 200 g for 10 minutes. Make sure that the cells have pelleted. If not, re-centrifuge for 5 minutes.  Place the tube upright on ice for 60 min.  Layer super over 15 ml of Ficoll-Hypaque leaving 7.5 ml of fluid above the pellet.  Resuspend the pellet by rotating the tube along the long axis.  Stand upright for 1 minute. Remove the top 5 ml and layer on Ficoll-Hypaque.  Rotate as above and transfer to gradient tube.  Wash the tube with 5 ml of PBS and add to gradient.  Centrifuge at 300 g for 40 minutes, room temperature.  Collect the B cells at the interface. Wash 3 times with PBS.  Suspend the SRBC-T cell pellet. Centrifuge at 300 d for 10 minutes.  Aspirate all of the supe. Break up the cell pellet by gently shaking.  Add 9 ml of di-H2O. with shaking for 4 seconds.  Add 1 ml of 10X PBS with shaking.  Immediately fill the tube with 1X PBS.  Centrifuge at 300 g for 10 minutes, and wash 2 times with PBS.